1. Field of the Invention
The present invention relates to an novel purified human activin. More specifically, the present invention relates to a human activin with an improved purity and a process for producing the same, particularly a process to isolate and purify human activin A from crude human activin A. Furthermore a highly purified human activin which can be obtained by such a process, and the human activin which is in a form of drug product.
2. Description of the Related Art
It is desirable to develop human activin as a medicine. Particularly, human activin A is a useful substance for the amelioration or treatment of osteoporosis or other use.
Human activin A is a homo-dimer protein consisting of two polypeptide chains of 116 amino acid residues respectively, which is isolated and purified from the culture supernatant of human leukemia cell line THP-1 (IFO 50147). The molecular weight of human activin A is about 25,000 daltons, 9 cysteine residues (Cys) exist in every polypeptide chain (total 18 residues in dimer), and total 9 disulfide bonds are formed intra- and intermolecularly (refer to Biochemical and Biophysical Research Communications, 142, 1095-1103, 1987).
The present inventors, in particular, have been developing the following four methods for the production and purification of human activin A. That is:
(1) a method for obtaining human activin A by ammonium sulfate fractionation and 4 steps of column chromatography from the culture supernatant of human leukemia cell line THP-1 (IFO 50147) after stimulating with phorbol ester (refer to Cell Technology, a separate volume 4, p.48-58, 1987);
(2) a method for obtaining human activin A by the combination of acid-organic solvent precipitation/cooling phase separation, and reverse-phase HPLC, from the culture supernatant of recombinant a CHO cell which is overproducing human activin A, obtained by introducing the expression vector in which the human activin cDNA was integrated (refer to Biochemical and Biophysical Research Communications, 151, 230-235, 1988; and Japanese Patent Kokai Publication JP-A-01-300898);
(3) a method for obtaining human activin A by affinity chromatography using follistatin, a human activin A binding protein as a ligand, from the culture supernatant obtained by the same method as described above (refer to Japanese Patent Kokai Publication JP-A-02-255098), and
(4) a method for obtaining human activin A by reverse-phase HPLC from crude human activin A solution which is obtained by solubilizing and refolding an inclusion body accumulated in the cell of recombinant microorganisms overproducing human activin A, to which the expression vector integrated human activin A CDNA was introduced (refer to WO97/23638).
The human activin A obtained by the four methods described above was pure enough for animal experimentation. However, for developing human activin A as a drug product, the practical purification process which can produce extremely high purity bulk of the drug product for pharmaceutical use in humans with industrial scale and appropriate production cost, has to be constructed. It was difficult to purify human activin A with enough purity for injecting to humans in the methods described above. Namely, it was impossible to remove completely an antigenic substance and a pyrogen etc. derived from a host or a medium, molecular variants based on the translational mistake or inappropriate post-translational processing of human activin A gene, and degradation, modification and the like products of human activin A produced in the purification process, even if the chromatography used in the four production methods described above may be combined in any way.
In view of these situations, a process for producing a highly purified human activin is desirable.
It is a problem to be solved by the present invention to develop a process in which a highly pure human activin A appropriate for pharmaceutical use from crude human activin, particularly crude human activin A, can be isolated and purified easily. Moreover, the activin can be produced on an industrial scale as highly pure human activin.
The present inventors have studied eagerly to solve the problem described above and have investigated crude human activin A as a crude human activin thoroughly.
It is considered to be most preferable to establish an ion exchange chromatography by using the difference in electrostatic character between human activin A and its variants to separate the human activin A from the crude human activin A solution obtained by refolding the inclusion body in the culture of recombinant microorganisms overproducing human activin A. For this reason, it is thought that the ion exchange chromatography is easy for up-scaling compared to reverse-phase chromatography and it is most suitable method for industrial large scale production of protein.
Eto et al. purified crude human activin A by anion exchange chromatography using two carriers such as DEAE-toyopearl (TOSOH Corp.) and Mono-Q (Amersham Pharmacia Biotech Limited) (refer to Biochemical and Biophysical Research Communications, 151, 230-235, 1988). However, when DEAE-toyopearl which is a packing material of column for large scale purification was used for purification, protein purity of human activin A thus obtained is low, ca. 2%. Even if Mono-Q, which is a column for high performance liquid chromatography (HPLC) with higher efficiency, was used, the protein purity was up to ca. 55%. Either method was not sufficient to get the purified protein appropriate for pharmaceutical use. In addition, when either column was used, the recovery of human activin A was low (ca. 56% for the former recovery, and ca. 63% for the latter recovery), and it was decided that these columns could not be used for industrial production as they were, because the recovered solution was very dilute. It was quite clear that the purification of human activin A by an ion exchange chromatography using conventional purification techniques in the industrial scale as described above, was extremely difficult.
In order to solve the above mentioned problem, the present inventors have proceeded the research and have found a method to purify human activin A to that of high purity from a crude human activin A solution by using the purification process involving a cation exchange chromatography, after removing low molecular weight impurities therefrom, if necessary, and finally completed the present invention based on these findings.
More precisely, according to the present invention, a highly pure human activin A can be obtained by the process which comprises removing the refolding agents in the crude human activin, especially the crude human activin A solution by the standard method, if they remain and it necessary, applying it (preferably containing a high concentration of organic solvent) to a cation exchange column equilibrated with the buffer solution of chaotropic ion and very low pH, and separating and removing a variant different in electrostatic character from the activin by concentration gradient elution method of chaotropic ion, with high efficiency.
Namely, when a microorganism to which the human activin A gene is introduced, is cultivated, and an active and a crude human activin A solution obtained by solubilizing and refolding the inclusion body of the human activin A thus produced is used for purification, by applying such solution to a cation exchange chromatography preferably in the condition combined with a high concentration of organic solvent, extremely low pH value, and a salt of chaotropic ion character, preferably after removing low molecular weight impurities therefrom, performing a chaotropic ion concentration gradient elution therefor, it is possible to remove effectively, more preferably, any impurities of a protein derived from a host, a non-refolded aggregate and a variant different in electrostatic character from the activin, and moreover obtain a concentrated human activin A with high recovering yield.
That is, the present invention is directed to a process for producing human activin with an improved purity, which comprises subjecting a crude human activin to a purification procedure involving a cation exchange chromatography, in particular, a cation exchange chromatography by a concentration gradient elution method. For the concentration gradient elution method, a chaotropic ion concentration gradient elution method is preferable.
As a representative example of human activin, human activin A can be exemplified.
Furthermore, the present invention contains the following embodiments:
[1] The process according to the process for the production described above, wherein the cation exchange chromatography is performed in the presence of water soluble organic solvent and/or in the acid condition.
[2] The process according to the process for the production described above [1], wherein in the purification method, the organic solvent contains at least one member selected from the group consisting of lower alcohol, acetonitrile, dimethyl sulfoxide and dimethylformamide, and its concentration is at least 20 volume %, more preferably 20 to 60 volume %.
[3] The process according to the process for the production described above, wherein the crude human activin contains at least one member selected from the group consisting of a variant different in electrostatic character from the activin, a protein derived from a host, an antigenic substance and a pyrogen etc. derived from a host and/or a medium, non-refolded aggregates in case of refolding, molecular variants based on the translational mistake and/or inappropriate post-translational processing of the gene (human activin A), and degradation, modification and the like products of human activin produced in the purification process.
[4] The process according to the process for the production described above, wherein the crude human activin contains at least one species in the impurities derived from an active and a crude human activin obtained by solubilizing and refolding an inclusion body of human activin produced by cultivating a microorganism to which the human activin A gene is introduced.
[5] The process according to the process for the production described above, wherein purification process for removing low molecular weight impurities is combined thereto as a purification process.
[6] The process for the production described above, involving further purification process by anion exchange chromatography under the alkaline condition.
[7] The process according to the process for the production described above [6], wherein the chromatography contains an anion exchange chromatography having natural polysaccharide as a base matrix.
[8] A process for producing a concentrated human activin at a high recovering yield, which comprises cultivating a microorganism to which the human activin gene is introduced, solubilizing and refolding the inclusion body of the human activin in thus produced, removing low molecular weight impurities from the active and crude human activin solution obtained above, applying this to a cation exchange chromatography equilibrated with the solution of extremely low pH value containing high concentration of organic solvent and chaotropic ion, and performing the chaotropic ion concentration gradient elution to remove a protein derived from a host, a non-refolded aggregate and a variant different in electrostatic character from the activin are removed effectively and highly.
[9] In the process for the production described above [8], the process for producing human activin with high purity, from the crude human activin solution, which comprises removing low molecular weight impurities from the solution, and combining the following 2 steps to purify it more effectively:
(i) A step for recovering concentrated human activin A quantitatively, which comprises applying said partially purified product to an anion exchange chromatography having natural polysaccharide as a base matrix, equilibrated under the extremely high pH condition, and substituting the buffer solution of higher salt concentration and lower pH value, added with organic solvents therefor, to remove almost all of a protein derived from a host, a non-refolded aggregate and a variant different in electrostatic character from the activin;
(ii) A step for obtaining concentrated human activin A with high recovering yield, which comprises substituting for the buffer solution of the fraction recovered in the above process (i), applying it to an anion exchange chromatography equilibrated with extremely high pH solution containing organic solvent, and performing an elution with the salt concentration gradient and pH gradient (lowering pH) to remove almost all of a protein derived from a host and a variant different in electrostatic character from the activin, followed by the step for the purification process of the above described cation exchange chromatography, after substituting for the buffer solution of the recovered fraction obtained in the above (ii).
[10] A human activin with improved purity and a highly purified human activin having a purity of at least 99%, obtained by the process for the production of the present invention and that of [1] to [9] described above.
[11] A human activin having a purity of at least 99%, which contains substantially no variant different in electrostatic characters from the activin, as impurity.
[12] In the processes for the production of the present invention and that of [1] to [9] described above, as well as those of [10] and [11] described above, the inventions wherein the human activin is a human activin A.
[13] An agent for amelioration or treatment of osteoporosis or other drug products comprising the human activin with improved purity, in particular, such human activin A, which is the present invention or which is obtained or can be obtained by the present invention.